ABSTRACT
AIM:To explore the role of nuclear factor-κB(NF-κB)and activator protein-1(AP-1)signaling pathway in the inhibitory effects of Agkistrodon acutivirus protein C activator(PCA)on lipopolysaccharide(LPS)-induced tissue factor(TF)expression in human umbilical vein endothelial cells(HUVECs).METHODS: The viability of the HUVECs was measured by MTT assay.The protein distribution of tumor necrosis factor-associated factor 6(TRAF6)in the cells was detected by immunohistochemical staining.The protein expression of NF-κB p65,TF,c-Fos and c-Jun was deter-mined by Western blot.The mRNA expression of TF in the HUVECs was detected by qPCR.The content of TF in the me-dium of each group was measured by ELISA.RESULTS:Compared with the control group,the viability of the HUVECs in LPS group decreased significantly(P<0.01), obvious yellow dye particles appeared in the cytoplasm, cytoplasmic stai-ning deepened,and the average absorbance of TRAF6 was increased(P<0.01).The protein expression of NF-κB p65, c-Jun and c-Fos were significantly increased(P<0.01).The expression of TF at mRNA and protein levels were signifi-cantly increased(P<0.01).Compared with the LPS group,the cell viability in PCA +LPS group was slightly increased (P<0.05),the cell morphology was normal,cytoplasmic yellow dye particles were not obvious, and the average absor-bance of TRAF6 was significantly lower than that in LPS group(P<0.01).The protein expression of NF-κB, c-Jun and c-Fos was significantly decreased(P<0.01),and the expression of TF at mRNA and protein levels were decreased(P<0.01).CONCLUSION:PCA significantly reduces the damage of HUVECs induced by LPS.The mechanism may be a-chieved by reducing the activation of TRAF 6,NF-κB and AP-1 nuclear transcription factors,thereby reducing the release of tissue factor.
ABSTRACT
AIM: To investigate the effects of protein C activator (PCA) from Agkistrondon acutus venom ( AAV) on the tension of thoracic aorta rings isolated from the rats with sepsis.METHODS:The model of sepsis was es-tablished by intraperitoneal injection of lipopolysaccharide ( LPS) .SD rats were randomly divided to 6 groups ( n=6 ):sham group, LPS group, PCA intervention group (LPS+PCA, PCA at doses of 0.1 mg/kg, 0.3 mg/kg and 0.6 mg/kg) and LPS+polymyxin B (at dose of 0.2 mg/kg) group.Using perfusion experiment in vitro, the tension of the aortic rings was measured by biological signal analytical system.RESULTS:The values of MABP, HR, LVDP and ±dp/dtmax were significantly lower in LPS group than those in sham group and LPS+PCA groups.Compared with sham group, the relaxa-tion response to acetylcholine ( ACh) and the contractile response of aorta rings induced by phenylephrine ( Phe) were sig-nificantly decreased in LPS group, which were increased significantly in PCA intervention group ( especially at dose of 0.6 mg/kg) compared with LPS group.The dose-response curve of aorta contraction with denuded endothelium induced by Phe shifted down significantly in LPS group compared with sham group, and no significant difference between LPS group and PCA intervention group was observed.Also no statistical difference was found in non-endothelium dependent relaxation of aortic rings induced by sodium nitroprusside among the groups.Pretreatment of N-nitro-L-arginine methl ester and methyl-ene blue increased the contraction amplitude of aortic rings induced by Phe.CONCLUSION:PCA from AAV effectively reverses the hypoergia of the vessels in rats with sepsis through protecting vascular endothelium, the mechanism of which may be mediated by inhibiting NO-GC-cGMP signal transduction pathway.